Carbohydrate Binding Protein 35 (=galectin-3) is a galatose-specific lectin found in a variety of cell and tissues. The protein is found in both the cytoplasm and nucleus of cells. Using a cell-free assay for the splicing of pre-mRNA, it has been demonstrated that nuclear extracts depleted of galectin-3 concomitantly loses splicing activity. The activity can be reconstituted by the addition of purified recombinant galectin-3, thus identifying the protein as a required splicing factor. When extracts of mouse 3T3 fibroblasts were subjected to two-dimensional gel electrophoresis and immunoblotting, two spots were observed, corresponding to pI values of 8.7 and 8.2. The pI 8.2 species represents post-translational modification of the galectin-3 polypeptide (pI 8.7) by the addition of a single phosphate group. The phosphorylated (pI 8.2) form is found in both the cytosolic and nuclear fractions, whereas the unphosphorylated (pI 8.7) species is found exclusively in the nuclei. Quiescent populations of 3T3 cells are characterized by the predominance of phosphorylated galectin-3. Stimulation of the same cells into the proliferative state resulted in an increase in the amount of phosphorylated species; more dramatic, however, is the elevation of the level of the unphosphorylated form, which is exclusively nuclear. On the basis of the observations, it is apparent that the physiological state of the cell regulates the phosphorylation state of galectin-3 and its nuclear versus cytoplasmic distribution. To this end, MALDI-MS analysis will be used to identify the amino acid residue that is phosphorylated, which in turn might provide insight into the type of kinase involved in regulation.